Cc-chemokine mutants against liver diseases

ABSTRACT

CC-Chemokine mutants having reduced Glycosaminoglycans (GAG)-binding properties are effective against liver fibrotic inflammatory and/or autoimmune diseases. Particularly preferred are the mutants of CCL5/RANTES having reduced GAG-binding properties.

FIELD OF THE INVENTION

The present invention relates to novel therapeutic applications ofCC-chemokine mutants having a reduced GAG-binding activity.

BACKGROUND OF THE INVENTION

Chemokines are secreted pro-inflammatory proteins of small dimensions(70-130 amino acids) mostly involved in the directional migration andactivation of cells, especially the extravasation of leukocytes from theblood to tissue localizations needing the recruitment of these cells(Baggiolini M et al., 1997; Rossi D and Ziotnik A, 2000; Fernandez E Jand Lolis E, 2002). Usually chemokines are produced at the site of aninjury, inflammation, or other tissue alteration in a paracrine orautocrine fashion, triggering cell-type specific migration andactivation.

Depending on the number and the position of the conserved cysteines inthe sequence, chemokines are classified into C-, CC-, CXC- andCX₃C-chemokines. Inside each of these families, chemokines can befurther grouped according to the homology of the entire sequence, or ofspecific segments.

A series of heptahelical G-protein coupled membrane receptors, are thebinding partners that allow chemokines to exert their biologicalactivity on the target cells, which present specific combinations ofreceptors according to their state and/or type. An unified nomenclaturefor chemokine ligands and receptors, which were originally named by thescientists discovering them in a very heterogeneous manner, has beenproposed to associate each of these molecule to a systemic nameincluding a progressive number CCL1, CCL2, etc. for CC-chemokines; CCR1,CCR2, etc. for CC-chemokines receptors, and so on.

The physiological effects of chemokines result from a complex andintegrated system of concurrent interactions. The receptors often haveoverlapping ligand specificity, so that a single receptor can binddifferent chemokines, as well a single chemokine can bind differentreceptors. In particular, N-terminal domain of chemokines is involved inreceptor binding and N-terminal processing can either activatechemokines or render chemokines completely inactive.

Amongst all the chemokines characterized so far, CC-chemokines, such asCCL5 (also known as RANTES; Appay V and Rowland-Jones S L, 2001) or CCL3(also known as MIP-1alpha, U.S. Pat. No. 6,355,476), have beenintensively studied to identify therapeutically useful molecules.Variants of CC-chemokines, missing up to nine N-terminal amino acids,have been tested for their activity as inhibitors or antagonists of thenaturally occurring forms. These molecules are inactive on monocytes andare useful as receptor antagonists (Gong J H et al., 1996; WO 99/16877).Alternatively, N-terminal extension of the mature CC-chemokine with oneMethionine results in almost complete inactivation of the molecule,which also behaves as an antagonist for the authentic one (WO 96/17935).

Moreover, in order to perform structure-function analysis ofCC-chemokines, variants containing substitutions or chemicalmodifications in different internal positions, as well as CC-chemokinederived peptides, have been tested for the interactions with receptorsor other molecules. Some of these variants have been disclosed as havingsignificatively altered binding properties, and sometimes they areactive as CC-chemokine antagonists, having potential therapeuticapplications in the treatment of HIV infection and some inflammatory orallergic diseases (WO 99/33989; Nardese V et al., 2001). In particular,the binding determinants and the physiological relevance of theinteractions of chemokines, by the means of specifically positionedbasic residues, with Glycosaminoglycans (GAGs) has been intensivelystudied (WO 02/28419; Vives R et al., 2002; McCornack M A et al., 2003;Stringer S E et al., 2002; Fukui S et al., 2002; Laurence J S et al.,2001; Martin L et al., 2001; Koopmann W and Krangel M S, 1997).

Even though there are potential drawbacks in using chemokines astherapeutic agents (tendency to aggregate, promiscuous binding), thesemolecules offer the possibility for therapeutic intervention inpathological conditions associated to such processes, in particular byinhibiting/antagonizing specific chemokines and their receptors at thescope to preventing the excessive recruitment and activation of cells,in particular leukocytes, for a variety of indications related toinflammatory and autoimmune diseases, cancers, and bacterial or viralinfections (Schneider G P et al., 2001, Baggiolini M, 2001; Godessart Nand Kunkel S L, 2001).

The possible therapeutic applications of chemokine-related compoundsagainst hepatic diseases have been intensively studied, as recentlyreviewed (Ajuebor M N et al., 2002; Marra F, 2002, Colletti L M, 1999).In particular, liver specific inflammation is mediated by activatedCD4(+) T cells and driven by an upregulation of the hepatic expressionof IFNgamma, but the mechanisms governing T cell migration from theblood into tissues during T cell-mediated hepatitis remains incompletelyunderstood, since the endogenous mediators that promote the recruitmentof T cells to the liver during T cell-mediated liver diseases have beenpoorly characterized.

It has been demonstrated that some chemokines are highly expressed andimportant for the recruitment for liver-infiltrating lymphocytes inhepatitis-related animal models (acetaminophen-induced, ConcanavalinA-induced, adenovirus-induced, or hepatitis B virus-specific),suggesting a specific role of these molecules in the development ofhepatitis (Bautista A P, 2002; Lalor P F et al., 2002; Hogaboam C M etal., 2000; Kusano F et al., 2000).

Some broad spectrum CC-chemokine antagonists were disclosed inconnection to hepatic diseases (WO 00/73327; WO01/58916; U.S. Pat. No.6,495,515). CXC chemokines are capable to induce rapid hepatocyteproliferation and liver regeneration after injury (WO 01/10899). CCR1 orMIP-1alpha antagonists can be used for inhibiting graft-related orischemia/reperfusion-related liver dysfunctions (WO 00/44365; Murai M etal., 1999).

However, prior art fails to describe any therapeutic efficacy of anisolated, specific CC-chemokine mutant generated by the substitution ofinternal residues, against liver fibrotic inflammatory and/or autoimmunediseases.

SUMMARY OF THE INVENTION

It has been surprisingly found that a CCL5/RANTES mutant having reducedGAG-binding properties, resulting from the substitution of specificinternal residues, counteracts liver fibrotic injury in a relevantanimal model.

These evidences demonstrate the possibility of using this and othermutants of CC-chemokines (such as CCL3/MIP-1alpha or CCL4/MIP-1beta)having similar reduced GAG-binding activity in the treatment of liverfibrotic inflammatory and/or autoimmune diseases. Particularly preferredare CCL5 mutants are the GAG-binding defective mutants of CCL5 generatedby appropriately mutagenising the GAG-binding domain of CCL5.

Other features and advantages of the invention will be apparent from thefollowing detailed description.

DESCRIPTION OF THE FIGURES

FIG. 1: effect of the treatment with a GAG-binding defective CCL5 mutant(triple 40's RANTES mutant) in an animal model for liver diseases basedon Concanavallin A (Con A) administration and measurement of serumalanine transaminase (ALT). The asterisk indicates the statisticalsignificance of the measured difference (P<0.05) when compared to thePBS-treated controls.

FIG. 2: time course analysis of the effect of Con A (Con A)administration on serum ALT (A) and on hepatic MIP-1 alpha (B) levels inmice.

FIG. 3: effect of the treatment with Con A or PBS on control (MIP-1α WT)and MIP-1alpha knock-out (MIP-1α KO) mice on serum ALT (A) or hepaticIFN-gamma (B) levels in mice. The levels were measured 8 hours after theadministration of PBS or Con A. The asterisk indicates the statisticalsignificance of the measured difference (P<0.05) when compared to thePBS-treated controls.

DETAILED DESCRIPTION OF THE INVENTION

The main object of the present invention is the use of a CC-chemokinemutant having a reduced GAG-binding activity for the treatment of liverfibrotic inflammatory and/or autoimmune diseases. In particular, suchmutants are the ones already disclosed in the prior art for theCC-chemokines CCL3/MIP-1alpha, CCL4/MIP-1beta, or CCL5/RANTES (WO02/28419; Laurence J S et al., 2001; Koopmann W and Krangel M S, 1997).

In particular, the CC-chemokine mutants have the sequence of the onesdisclosed in the prior art under the names of triple 40's RANTES mutant(SEQ ID NO: 1), triple MIP-1alpha mutant (SEQ ID NO: 2), and triple 40'sMIP-1beta mutant (SEQ ID NO: 3) mutants. It is however evident that anyother corresponding mutant of CCL3/MIP-1alpha, CCL4/MIP-1beta, orCCL5/RANTES having reduced GAG-binding properties resulting from thesubstitution of the same residues disclosed in the prior art but with adifferent amino acid (i.e. the basic residue is substituted with anon-polar amino acid other than Ala or with an acid residue), orresulting from a substitution in other position(s) can be used accordingto the invention.

These polypeptides can be prepared by chemical synthesis, bysite-directed mutagenesis techniques, or any other known techniquesuitable thereof, which provide a finite set of substantiallycorresponding mutated or shortened peptides or polypeptides which can beroutinely obtained and tested by one of ordinary skill in the art usingthe teachings presented in the prior art and in the Examples of thepresent patent application. Similar compounds may also result fromconventional mutagenesis technique of the encoding DNA, fromcombinatorial technologies at the level of encoding DNA sequence (suchas DNA shuffling, phage display/selection), or from computer-aideddesign studies based on the tridimensional structure and otherfunctional assays of chemokines, with or without the presence of GAGs(Rajarathnam K, 2002; Vives R et al., 2002; McCornack M A et al., 2003;Stringer S E et al., 2003; Fukui S et al., 2002; Martin L et al., 2001).

The above cited prior art on GAG-binding defective CC-chemokine mutantsfails to identify liver fibrotic inflammatory and/or autoimmune diseasesas therapeutic indications in which these molecules can provide abeneficial effect. As there are currently therapies only partiallyeffective and/or acceptable for treating diseases such as alcoholicliver diseases, viral or autoimmune hepatitis, the disclosedCC-chemokine mutants represent alternative therapeutic compoundspossibly better accepted and efficient than the current therapies

The wording “a reduced GAG-binding activity” or “GAG-binding defective”means that the CC-chemokine mutants have a lower ability to bind toGAGs, i.e. a lower percentage of each of these mutants bind to GAGs(like heparin sulphate) with respect to the corresponding wild-typemolecule, as measured with the assays in the above cited prior artdisclosing such mutants.

In addition to the mutation at the specific positions leading to thedecreased affinity for GAGs, the CC-chemokine mutants may include othermodifications with respect to the wild-type molecule, generating activevariants of said CC-chemokine mutants in which one or more amino acidshave been added, deleted, or substituted in a conservative manner. Theseadditional modifications should be intended to maintain, or evenimprove, the properties of the specific mutants, or by any otherrelevant means known in the art, making them equally useful for treatingliver fibrotic inflammatory and/or autoimmune diseases. Other additionalpreferred changes in these active variants are commonly known as“conservative” or “safe” substitutions, that is, with amino acids havingsufficiently similar chemical properties, in order to maintain thestructure and the biological function of the CC-chemokine mutant. It isclear that insertions and deletions of amino acids may also be made inthe above defined sequences without altering their function,particularly if the insertions or deletions only involve a few aminoacids, e.g., under ten, and preferably under three, and do not remove ordisplace amino acids which are critical to the functional conformationof a protein or a peptide.

The literature provide many models on which the selection ofconservative amino acids substitutions can be performed on the basis ofstatistical and physico-chemical studies on the sequence and/or thestructure of natural protein (Rogov S I and Nekrasov A N, 2001). Proteindesign experiments have shown that the use of specific subsets of aminoacids can produce foldable and active proteins, helping in theclassification of amino acid “synonymous” substitutions which can bemore easily accommodated in protein structure, and which can be used todetect functional and structural homologs and paralogs (Murphy L R etal., 2000). The synonymous amino acid groups and more preferredsynonymous groups are those defined in Table I.

Alternatively, active CC-chemokine mutants may contain on or morenon-natural, amino acid derivatives being “synonymous” to a naturalamino acid, are those defined in Table II. By “amino acid derivative” isintended an amino acid or amino acid-like chemical entity other than oneof the 20 genetically encoded naturally occurring amino acids. Inparticular, the amino acid derivative may contain substituted ornon-substituted alkyl linear, branched, or cyclic moieties, and mayinclude one or more heteroatoms. The amino acid derivatives can be madede novo or obtained from commercial sources (Calbiochem-Novabiochem AG,Switzerland; Bachem, USA). Various methodologies for incorporatingunnatural amino acids derivatives into proteins, using both in vitro andin vivo translation systems, to probe and/or improve protein structureand function are disclosed in the literature (Dougherty D A, 2000).

The term “active” means that such alternative compounds should maintainthe therapeutic properties of the CC-chemokines mutants against liverfibrotic inflammatory and/or autoimmune diseases as described in thepresent invention, and should be as well pharmaceutically acceptable anduseful.

In another embodiment, a polypeptide comprising the GAG-bindingdefective CC-chemokine mutant and an amino acid sequence belonging to aprotein sequence other than the corresponding CC-chemokine can be alsoused for treating liver fibrotic inflammatory and/or autoimmunediseases. The heterologous sequence is intended provide additionalproperties without considerably impairing the therapeutic activity.Examples of such additional properties are an easier purificationprocedure, a longer lasting half-life in body fluids, an additionalbinding moiety, the maturation by means of an endoproteolytic digestion,or extracellular localization. This latter feature is of particularimportance for defining a specific group of fusion or chimeric proteinsincluded in the above definition since it allows the CC-chemokinemutants to be localized in the space where not only where the isolationand purification of these polypeptides is facilitated, but also whereCC-chemokines naturally interact with receptors and other molecules.Design of the moieties, ligands, and linkers, as well methods andstrategies for the construction, purification, detection and use offusion proteins are widely discussed in the literature (Nilsson J etal., 1997; “Applications of chimeric genes and hybrid proteins” MethodsEnzymol. Vol. 326-328, Academic Press, 2000; WO 01/77137).

Additional protein sequences can be chosen amongst extracellular domainsof membrane-bound protein, immunoglobulin constant regions,multimerization domains, extracellular proteins, signalpeptide-containing proteins, export signal-containing proteins. Thechoice of one or more of these sequences to be fused to the CC-chemokinemutants is functional to the desired use, delivery and/or preparationmethod.

The GAG-binding defective CC-chemokine mutants can be also provided forthe treatment of liver fibrotic inflammatory and/or autoimmune diseasesin the form of the corresponding active precursor, salt, derivative,conjugate or complex. These alternative forms may be preferred accordingto the desired method of delivery and/or production.

The “precursors” are compounds which can be converted into othercompounds by metabolic and enzymatic processing prior or after theadministration to the cells or to the organism.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the peptides, polypeptides, oranalogs thereof. Salts of a carboxyl group may be formed by means knownin the art and include inorganic salts, for example, sodium, calcium,ammonium, ferric or zinc salts, and the like, and salts with organicbases as those formed, for example, with amines, such astriethanolamine, arginine or lysine, piperidine, procaine and the like.Acid addition salts include, for example, salts with mineral acids suchas, for example, hydrochloric acid or sulfuric acid, and salts withorganic acids such as, for example, acetic acid or oxalic acid. Any ofsuch salts should have substantially similar activity to the originalpolypeptide.

The term “fractions” as herein used refers to derivatives which can beprepared from the functional groups present on the lateral chains of theamino acid moieties or on the N-/ or C-terminal groups according toknown methods. Such derivatives include for example esters or aliphaticamides of the carboxyl-groups and N-acyl derivatives of free aminogroups or O-acyl derivatives of free hydroxyl-groups and are formed withacyl-groups as for example alcanoyl- or aroyl-groups. Alternatively, thederivatives may contain sugars or phosphates groups linked to thefunctional groups present on the lateral chains of the amino acidmoieties. Such molecules can result from in vivo or in vitro processeswhich do not normally alter primary sequence, for example chemicalderivativization of peptides (acetylation or carboxylation),phosphorylation (introduction of phosphotyrosine, phosphoserine, orphosphothreonine residues) or glycosylation (by exposing the peptide toenzymes which affect glycosylation e.g., mammalian glycosylating ordeglycosylating enzymes).

The term “derivatives” as herein used refers to derivatives which can beprepared from the functional groups present on the lateral chains of theamino acid moieties or on the N- or C-terminal groups according to knownmethods. Such derivatives include for example esters or aliphatic amidesof the carboxyl-groups and N-acyl derivatives of free amino groups orO-acyl derivatives of free hydroxyl-groups and are formed withacyl-groups as for example alcanoyl- or aroyl-groups.

Alternatively, useful conjugates or complexes of the CC-chemokinemutants can be generated by using molecules and methods known in the artfor improving the detection of the interaction with other proteins(radioactive or fluorescent labels, biotin), therapeutic efficacy(cytotoxic agents, isotopes), or drug delivery efficacy, such aspolyethylene glycol and other natural or synthetic polymers (Pillai Oand Panchagnula R, 2001). In the latter case, a site-directedmodification of an appropriate residue, present in the natural sequenceor introduced by mutating the natural sequence, at an internal orterminal position, can be introduced. Similar modifications have beenalready disclosed for chemokines (WO 02/04499; WO 02/04015; Vita C etal., 2002).

Any residue can be used for attachment, provided it has a side-chainamenable for polymer attachment (i.e., the side chain of an amino acidbearing a functional group, e.g., lysine, aspartic acid, glutamic acid,cysteine, histidine, etc.). Alternatively, a residue at these sites canbe replaced with a different amino acid having a side chain amenable forpolymer attachment. Polymers suitable for these purposes arebiocompatible, namely, they are non-toxic to biological systems, andmany such polymers are known. Such polymers may be hydrophobic orhydrophilic in nature, biodegradable, non-biodegradable, or acombination thereof. These polymers include natural polymers (such ascollagen, gelatin, cellulose, hyaluronic acid), as well as syntheticpolymers (such as polyesters, polyorthoesters, polyanhydrides). Examplesof hydrophobic non-degradable polymers include polydimethyl siloxanes,polyurethanes, polytetrafluoroethylenes, polyethylenes, polyvinylchlorides, and polymethyl methaerylates. Examples of hydrophilicnon-degradable polymers include poly(2-hydroxyethyl methacrylate),polyvinyl alcohol, poly(N-vinyl pyrrolidone), polyalkylenes,polyacrylamide, and copolymers thereof. Preferred polymers comprise as asequential repeat unit ethylene oxide, such as polyethylene glycol(PEG).

The preferred method of attachment employs a combination of peptidesynthesis and chemical ligation. Advantageously, the attachment of awater-soluble polymer will be through a biodegradable linker, especiallyat the N-terminal region of a protein. Such modification acts to providethe protein in a “pro-drug” form that, upon degradation of the linker,releases the protein without polymer modification.

The GAG-binding defective CC-chemokine mutants may be prepared by anyappropriate procedure in the art, such as recombinant DNA-relatedtechnologies involving the expression in Eukaryotic cells (e.g. yeasts,insect or mammalian cells) or Prokaryotic cells. Detailed methods forproducing the GAG-binding defective CC-chemokine mutants can be found inthe prior art originally disclosing them (WO 02/28419; Laurence J S etal., 2001; Koopmann W and Krangel M S, 1997), as well as in otherliterature featuring protocols for chemokine production (Edgerton M D etal., 2000) or common molecular biology techniques for the production ofrecombinant proteins in Prokaryotic or Eukaryotic host cells, such assome titles in the series “A Practical Approach” published by OxfordUniversity Press (“DNA Cloning 2: Expression Systems”, 1995; “DNACloning 4: Mammalian Systems”, 1996; “Protein Expression”, 1999;“Protein Purification Techniques”, 2001).

Alternatively the GAG-binding defective CC-chemokine mutants may beprepared by any other well known procedure in the art, in particular, bythe well established chemical synthesis procedures, which can beefficiently applied on these molecule given the short length. Totallysynthetic chemokines, also containing additional chemical groups, aredisclosed in the literature (Brown A et al., 1996; Vita C et al., 2002).

Examples of chemical synthesis technologies are solid phase synthesisand liquid phase synthesis. As a solid phase synthesis, for example, theamino acid corresponding to the N-terminus of the peptide to besynthetized is bound to a support which is insoluble in organicsolvents, and by alternate repetition of reactions, one wherein aminoacids with their amino groups and side chain functional groups protectedwith appropriate protective groups are condensed one by one in orderfrom the C-terminus to the N-terminus, and one where the amino acidsbound to the resin or the protective group of the amino groups of thepeptides are released, the peptide chain is thus extended in thismanner. Solid phase synthesis methods are largely classified by the tBocmethod and the Fmoc method, depending on the type of protective groupused. Typically used protective groups include tBoc (t-butoxycarbonyl),Cl-Z (2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloxycarbonyl), Bzl(benzyl), Fmoc (9-fluorenylmethoxycarbonyl), Mbh(4,4′-dimethoxydibenzhydryl), Mtr(4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl),Z (benzyloxycarbonyl) and Cl2-Bzl (2,6-dichlorobenzyl) for the aminogroups; NO2 (nitro) and Pmc (2,2,5,7,8-pentamethylchromane-6-sulphonyl)for the guanidino groups); and tBu (t-butyl) for the hydroxyl groups).After the synthesis, the desired peptide is subjected to thede-protection reaction and cut out from the solid support. Such peptidecutting reaction may be carried with hydrogen fluoride ortri-fluoromethane sulfonic acid for the Boc method, and with TFA for theFmoc method. Finally, the intact full-length peptides are purified andchemically or enzymatically folded (including the formation ofdisulphide bridges between cysteines) into the correspondingCC-chemokine mutants.

Purification of the natural, synthetic or recombinant proteins iscarried out by any one of the methods known for this purpose, i.e. anyconventional procedure involving extraction, precipitation,chromatography, electrophoresis, or the like. A further purificationprocedure that may be used in preference for purifying the protein isaffinity chromatography using monoclonal antibodies, heparin, or anyother suitable ligand that can bind the target protein at highefficiency and can be immobilized on a gel matrix contained within acolumn. Impure preparations containing the proteins are passed throughthe column. The protein will be bound to the column by means of thisligand while the impurities will pass through. After washing, theprotein is eluted from the gel by a change in pH or ionic strength.Alternatively, HPLC (High Performance Liquid Chromatography) can be alsoused.

Another object of the present invention is the use of a CC-chemokinemutant, wherein the CC-chemokine is CCL3/MIP-1alpha, CCL4/MIP-1beta, orCCL5/RANTES, having reduced GAG-binding activity in the preparation of apharmaceutical composition for liver inflammatory and/or fibroticdiseases, in particular when formulated in combination withpharmaceutically acceptable carriers, excipients, stabilizers,adjuvants, or diluents.

A non-limitative list of disorders involving hepatic damage in which theCC-chemokine mutant having reduced GAG-binding activity can be usedincludes alcoholic liver diseases (cirrohosis, steatosis), a viralhepatitis, an autoimmune hepatitis, or any other liver fibroticdegeneration.

Still another object of the present invention are methods for thetreatment or prevention of a liver inflammatory and/or fibrotic disease,comprising the administration of an effective amount of a CC-chemokinemutant having reduced GAG-binding activity, wherein the CC-chemokine isCCL3/MIP-1 alpha, CCL4/MIP-1 beta, or CCL5/RANTES.

The CC-chemokine mutants may be used alone, or with another therapeuticcomposition acting synergically or in a coordinated/sequential mannerwith them in the treatment of liver inflammatory and/or fibroticdiseases. For example, similar synergistic properties of CC-chemokinemutants have been demonstrated in combination with cyclosporin (WO00/16796).

In view of the claimed uses and methods of treatment, any drug deliverymethod allowing the targeting of the GAG-binding defective CC-chemokinemutant is preferred. Similar methods are known in the prior and mayinvolve the conjugation of the CC-chemokine mutant with galactosylatedor mannosylated albumin (Chuang V T et al., 2002) or the synthesis ofpolymeric nanoparticles from a sugar-containing conjugate composed oflactobionic acid, diamine-terminated polyethylene glycol) and cholicacid (Kim I S and Kim S H, 2002).

An “effective amount” refers to an amount of the active ingredients thatis sufficient to affect the course and the severity of the disease,leading to the reduction or remission of the liver pathology. Theeffective amount will depend on the route of administration and thecondition of the patient.

The pharmaceutical compositions may be formulated in any acceptable wayto meet the needs of the mode of administration for treating liverdiseases. For example, the use of biomaterials and other polymers fordrug delivery, as well the different techniques and models to validate aspecific mode of administration, are disclosed in literature (Luo B andPrestwich G D, 2001; Cleland J L et al., 2001).

“Pharmaceutically acceptable” is meant to encompass any carrier, whichdoes not interfere with the effectiveness of the biological activity ofthe active ingredient and that is not toxic to the host to which isadministered. Carriers can be selected also from starch, cellulose,talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, magnesium stearate, sodium stearate, glycerol monostearate,sodium chloride, dried skim milk, glycerol, propylene glycol, water,ethanol, and the various oils, including those of petroleum, animal,vegetable or synthetic origin (peanut oil, soy bean oil, mineral oil,sesame oil). For example, for parenteral administration, the aboveactive ingredients may be formulated in unit dosage form for injectionin vehicles such as saline, dextrose solution, serum albumin andRinger's solution.

Besides the pharmaceutically acceptable carrier, the compositions of theinvention can also comprise minor amounts of additives, such asstabilizers, excipients, buffers and preservatives that may facilitatethe processing of the active compounds into preparations which can beused pharmaceutically. Moreover, these compositions may contain anotheractive ingredient that can act synergically or in a coordinated mannerwith the CC-chemokine mutants.

The administration of such active ingredient may be by intravenous,intramuscular or subcutaneous route. Other routes of administration,which may establish the desired effects of the respective ingredients inthe liver, are comprised by the present invention. For example,administration may be by various parenteral routes such as subcutaneous,intravenous, intradermal, intramuscular, intraperitoneal, intranasal,transdermal, oral, or buccal routes. The pharmaceutical compositions ofthe present invention can also be administered in sustained orcontrolled release dosage forms, including depot injections, osmoticpumps, and the like, for the prolonged administration of the polypeptideat a predetermined rate, preferably in unit dosage forms suitable forsingle administration of precise dosages.

Parenteral administration can be by bolus injection or by gradualperfusion over time. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions,which may contain auxiliary agents or excipients known in the art, andcan be prepared according to routine methods. In addition, suspension ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions that may containsubstances increasing the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers. Pharmaceuticalcompositions include suitable solutions for administration by injection,and contain from about 0.01 to 99.99 percent, preferably from about 20to 75 percent of active compound together with the excipient.

The optimal dose of active ingredient may be appropriately selectedaccording to the route of administration, patient conditions andcharacteristics (sex, age, body weight, health, size), extent ofsymptoms, concurrent treatments, frequency of treatment and the effectdesired. Adjustment and manipulation of established dosage ranges arewell within the ability of those skilled.

Usually a daily dosage of active ingredient can be about 0.01 to 100milligrams per kilogram of body weight. Ordinarily 1 to 40 milligramsper kilogram per day given in divided doses or in sustained release formis effective to obtain the desired results. Second or subsequentadministrations can be performed at a dosage, which is the same, lessthan, or greater than the initial or previous dose administered to theindividual.

The present invention has been described with reference to the specificembodiments, but the content of the description comprises allmodifications and substitutions, that can be brought by a person skilledin the art without extending beyond the meaning and purpose of theclaims.

The invention will now be described by means of the following Examples,which should not be construed as in any way limiting the presentinvention. The Examples will refer to the Figures specified here below.

EXAMPLES Example 1 Efficacy of Different CCL5 Mutant Having a ReducedGAG-Binding Activity in a Hepatitis Model

Concanavalin A (Con A)-induced liver injury depends on the activation ofrecruitment of CD4(+) T cells by macrophages. In general, T cells arethe driving force underlying immunologically mediated hepatic disorders,making therefore the Con-A model highly relevant for studying thepathophysiology of diseases such as autoimmune or acute hepatitis(Takeda K et al., 2000; Tiegs G et al., 1992).

The assay involves the measurement of serum alanine aminotransferase(ALT), a liver enzyme contained in hepatocytes and released into serumwhen these cells are damaged. ALT is the most widely used marker inhumans and animals to document damage and destruction of liver cells (asin hepatitis), and, generally, ALT concentration correlates withhistological changes.

As example of CC-chemokine mutant having a reduced GAG-binding activity,the CCL5 mutant called triple 40's RANTES mutant was chosen andexpressed in E. coli and purified as previously described (WO 02/28419).

Specific pathogen-free male C57BL/6 mice (body weight of 21-23 grams;Charles River Breeding Farms) were treated with Phosphate Buffer Saline(PBS; vehicle control; 0.1 ml), or with one of above described CCL5mutants (30 micrograms/mouse in 0.1 ml PBS). The mice (5-10 per group)were injected s.c. 1 hour prior to Con-A i.v. injection (freshlyprepared Con A type V, 0.25 mg/mouse in 0.1 ml PBS; Sigma). At 8 hoursafter Con A administration and under halothane anaesthesia, blood wascollected for measuring plasma ALT using a commercial kit for ALTdetermination (Sigma).

This biochemical approach for quantifying liver injury showed that thetriple 40's RANTES mutant is capable of reducing ALT concentration inserum of the pre-treated animals in a statistically significant manner(FIG. 1).

Example 2 Relevance of MIP-1Alpha Expression in the Concanavallin aMouse Models

A time course experiments on normal mice was performed to verify ifthere is any correlation between ALT levels and hepatic CCL3/MIP-1 alphaprotein concentration in control male C57BL/6J mice and CCL3/MIP-1alphaknock-out C57BL/6J (Cook D N et al., 1995).

Mice (body weight of 21-23 grams; 5-6 weeks old; commercially availablethrough Jackson Laboratories and Charles River Breeding Farms) wereinjected intravenously with freshly prepared Concanavallin A (Con A;13.5 mg/kg; Sigma) in 0.1 ml Phosphate Buffer Saline (PBS), or with 0.1PBS only (vehicle control).

The levels of serum alanine transaminase (ALT) were measured using acommercial kit (Sigma). Blood was collected from mice at 30 minutes, 90minutes, 8 hours and 24 hours after Con A administration and underhalothane anesthesia.

In a separate set of experiments, the levels of hepatic CCL3/MIP-1alphaor IFNgamma were determined in control and CCL3/MIP-1 alpha knock-outmice that were injected with Con A or PBS only. Mice were sacrificed atthe indicated time points after Con A or PBS injection and livers wereperfused with ice-cold sterile PBS to remove blood elements. Individualperfused livers were homogenised in ice-cold PBS buffer containingprotease cocktail inhibitor (Sigma) immediately after their removal,tissue homogenates were centrifuged twice, and the supernatants filteredthrough a 0.45-mm filter and stored at −80° C. until used for proteindetermination. Hepatic CCL3/MIP-1alpha and IFNgamma levels were measuredby specific murine ELISA following a published protocol (Bone-Larson C Let al., 2001), and using commercial antibodies (R&D Systems). Totalprotein concentration in the homogenates was calculated using acommercial protein colorimetric assay (Bio-Rad Laboratories).

The data demonstrated that maximum hepatic injury, as evaluated by usingALT concentration, and peak hepatic CCL3/MIP-1alpha levels can be bothobserved at 8 hours after intravenous administration of a single dose ofCon A (13.5 mg/kg). This compound caused a more than 100-fold increaseof ALT levels (FIG. 2A) paralleled by a more than five-fold increase ofhepatic CCL3/MIP-1alpha protein expression (FIG. 2B) above control PBSinjection.

The effect of Con A administration in transgenic mice lacking theCCL3/MIP-1alpha gene to verify if CCL3/MIP-1 alpha deficiency impairsthe development of Con A-induced hepatic injury. In fact,CCL3/MIP-1alpha knock-out mice exhibited significantly less hepaticinjury 8 hours after the Con A injection relative to control m ice, asdemonstrated biochemically by a significant reduction (approx.five-fold) in ALT level (FIG. 3A). Moreover, hepatic concentration ofIFNgamma, one of the cytokines implicated in the pathogenesis of ConA-induced hepatitis since it is produced by CD4(+) T cells recruited tothe liver (Mizuhara H et al., 1996), is significantly lowered inCCL3/MIP-1alpha knock-out mice at 8 hours after Con A administration,when compared to control mice (FIG. 3B).

All data are shown as Mean±Standard Deviation. For comparisons of meansbetween 2 experimental groups (n=4-10 mice per group) a Student'sunpaired t-test was used, considering P value <0.05 statisticallysignificant Statistical analyses were performed using GraphPad Instat(version 3.00) Software.

The analysis of the data generated with the substituted CCL5 mutant andin MIP-alpha knock-out mice demonstrate how, using a single CC-chemokinemutant having the specific binding profile characterized in the priorart (WO 02/28419; Laurence J S et al., 2001; Koopmann W and Krangel M S,1997), a surprising therapeutically relevant effect can be obtained,suggesting the use of similar molecules in the treatment of fibroticliver diseases involving inflammatory and/or autoimmunitary reactions.The comparison with results obtained using other animal models for suchdiseases (acetaminophen-induced or adenovirus-induced hepatitis), mayprovide further confirmation of the therapeutic applicability of thesespecific CC-chemokine mutants. TABLE I More Preferred Amino AcidSynonymous Group Synonymous Groups Ser Gly, Ala, Ser, Thr, Pro Thr, SerArg Asn, Lys, Gln, Arg, His Arg, Lys, His Leu Phe, Ile, Val, Leu, MetIle, Val, Leu, Met Pro Gly, Ala, Ser, Thr, Pro Pro Thr Gly, Ala, Ser,Thr, Pro Thr, Ser Ala Gly, Thr, Pro, Ala, Ser Gly, Ala Val Met, Phe,Ile, Leu, Val Met, Ile, Val, Leu Gly Ala, Thr, Pro, Ser, Gly Gly, AlaIle Phe, Ile, Val, Leu, Met Ile, Val, Leu, Met Phe Trp, Phe, Tyr Tyr,Phe Tyr Trp, Phe, Tyr Phe, Tyr Cys Ser, Thr, Cys Cys His Asn, Lys, Gln,Arg, His Arg, Lys, His Gln Glu, Asn, Asp, Gln Asn, Gln Asn Glu, Asn,Asp, Gln Asn, Gln Lys Asn, Lys, Gln, Arg, His Arg, Lys, His Asp Glu,Asn, Asp, Gln Asp, Glu Glu Glu, Asn, Asp, Gln Asp, Glu Met Phe, Ile,Val, Leu, Met Ile, Val, Leu, Met Trp Trp, Phe, Tyr Trp

TABLE II Amino Acid Synonymous Group Ser D-Ser, Thr, D-Thr, allo-Thr,Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Arg D-Arg, Lys, D-Lys,homo-Arg, D-homo-Arg, Met, Ile, D-.Met, D-Ile, Orn, D-Orn Leu D-Leu,Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Pro D-Pro,L-I-thioazolidine-4-carboxylic acid, D-or L-1-oxazolidine-4-carboxylicacid Thr D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val,D-Val Ala D-Ala, Gly, Aib, B-Ala, Acp, L-Cys, D-Cys Val D-Val, Leu,D-Leu, Ile, D-Ile, Met, D-Met, AdaA, AdaG Gly Ala, D-Ala, Pro, D-Pro,Aib, .beta.-Ala, Acp Ile D-Ile, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met,D-Met Phe D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4,or 5-phenylproline, AdaA, AdaG, cis-3,4, or 5-phenylproline, Bpa, D-BpaTyr D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Cys D-Cys, S-Me-Cys, Met,D-Met, Thr, D-Thr Gln D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp AsnD-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Lys D-Lys, Arg, D-Arg,homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Asp D-Asp,D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Glu D-Glu, D-Asp, Asp, Asn, D-Asn,Gln, D-Gln Met D-Met, S--Me--Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val

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1-10. (canceled)
 11. A method for the treatment liver fibroticinflammatory, autoimmune diseases or liver fibrotic/autoimmune diseasecomprising the administration of an effective amount of a CC-chemokinemutant having reduced GAG-binding activity, wherein the CC-chemokine isCCL3/MIP-1alpha, CCL4/MIP-1beta, or CCL5/RANTES.
 12. The methodaccording to claim 11, wherein the CC-chemokine is CCL5/RANTES and themutant is triple 40's RANTES mutant (SEQ ID NO: 1).
 13. The methodaccording to claim 11, wherein the CC-chemokine is CCL3/MIP-1alpha andthe mutant is triple MIP-1alpha mutant (SEQ ID NO: 2).
 14. The methodaccording to claim 11, wherein the CC-chemokine is CCL4/MIP-1beta andthe mutant is triple MIP-1beta mutant (SEQ ID NO: 3).
 15. The methodaccording to claim 11, wherein the CC-chemokine mutant is an activevariant of said CC-chemokine mutant in which one or more amino acidshave been inserted, deleted, or substituted in a conservative manner.16. The method according to claim 12, wherein the CC-chemokine mutant isan active variant of said CC-chemokine mutant in which one or more aminoacids have been inserted, deleted, or substituted in a conservativemanner.
 17. The method according to claim 13, wherein the CC-chemokinemutant is an active variant of said CC-chemokine mutant in which one ormore amino acids have been inserted, deleted, or substituted in aconservative manner.
 18. The method according to claim 14, wherein theCC-chemokine mutant is an active variant of said CC-chemokine mutant inwhich one or more amino acids have been inserted, deleted, orsubstituted in a conservative manner.
 19. The method according to claim11, wherein the CC-chemokine mutant is an active variant of saidCC-chemokine mutant in which one or more amino acids have been inserted,deleted, or substituted in a conservative manner.
 20. The methodaccording to claim 11, wherein the CC-chemokine mutant further comprisesan amino acid sequence belonging to a protein sequence other than thecorresponding CC-chemokine.
 21. The method according to claim 11,wherein the CC-chemokine mutant is in the form of an active precursor,salt, derivative, conjugate or complex.
 22. The method according toclaim 11, wherein the liver disease is an alcoholic liver disease, aviral hepatitis, or an autoimmune hepatitis.